Indian J Microbiol (Jan–Mar 2011) 51(1):30–36 DOI 10.1007/s12088-011-0076-7
ORIGINAL ARTICLE
Biosurfactant Production by Pseudomonas aeruginosa from Renewable Resources R. Thavasi • V. R. M. Subramanyam Nambaru S. Jayalakshmi • T. Balasubramanian • Ibrahim M. Banat
•
Received: 20 August 2008 / Accepted: 29 May 2009 / Published online: 25 January 2011 Ó Association of Microbiologists of India 2011
Abstract This study deals with production and characterization of biosurfactant from renewable resources by Pseudomonas aeruginosa. Biosurfactant production was carried out in 3L fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (11.6 mg/ml) and biosurfactant production (8.6 mg/ml) occurred with peanut oil cake at 120 and 132 h respectively. Characterization of the biosurfactant revealed that, it is a lipopeptide with chemical composition of protein (50.2%) and lipid (49.8%). The biosurfactant (1 mg/ml) was able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene, comparatively the emulsification activity was higher than the activity found with Triton X-100 (1 mg/ml). Results obtained in the present study showed the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources. Emulsification activity found with the biosurfactant against different hydrocarbons showed its possible application in bioremediation of environments polluted with various hydrocarbons.
R. Thavasi (&) Department of Chemical and Biological Sciences, Polytechnic Institute of New York University, 6 Metrotech Center, Brooklyn, NY 11201, USA e-mail:
[email protected] V. R. M. Subramanyam Nambaru S. Jayalakshmi T. Balasubramanian CAS in Marine Biology, Annamalai University, Parangipettai, Chidambaram, Tamil Nadu 608 502, India I. M. Banat School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, Northern Ireland, UK
123
Keywords Biodegradation Bioremediation Biosurfactant Emulsification Lipopeptides
Introduction Many microorganisms produce extracellular or membrane associated surface-active compounds (biosurfactants). Biosurfactants are organic compounds belonging to various classes including glycolipids, lipopeptides, fatty acids, phospholipids, neutral lipids and lipopolysaccharides [1]. The properties/applications of biosurfactants includes excellent detergency, emulsification, foaming, dispersing traits, wetting, penetrating, thickening, microbial growth enhancement, metal sequestering and resource recovering (oil) which make surfactants replace some of the most versatile process chemicals [1]. Biosurfactants are promising natural surfactants that offer several advantages over chemically synthesized surfactants, such as lower toxicity, biodegradability and ecological acceptability. Although biosurfactants exhibit such important advantages, they have not been yet employed extensively in industry because of relatively high production cost. One possible strategy for reducing cost is the utilization of alternative substrates such as agro industrial wastes [2]. The main problem related to use of alternative substrates as culture medium is to find a waste with the right balance of nutrients that permits cell growth and product accumulation [3]. Molasses [4], peat hydrolysate [5] and potato process effluents [6] are examples of alternative substrates that have been suggested for biosurfactant production by Bacillus megaterium. The establishment of waste-based medium for biosurfactant production also faces another problem; once the kind and the properties of final product are depend on the composition of culture media [7].
Indian J Microbiol (Jan–Mar 2011) 51(1):30–36
Although biosurfactants have many interesting properties, their industrial importance depends on the ease of production [8]. Low yield of biosurfactant is a major limitation influencing its commercialization. Hence, in the present study peanut oil cake and waste motor lubricant oil were tried as cheaper carbon sources as compared to glucose and other petroleum based substrates for biosurfactant production. Peanut oil cake is a carbohydrate, protein and lipid rich residue generated at large amounts during the production of peanut oil, the cost of this cake is also very low when compared to other carbon sources like glucose, fructose, crude oil and other hydrocarbons. Waste motor lubricant oil is waste oil drained from geared motor vehicles after a long run, which contains weathered hydrocarbon fractions and may be useful for the biosurfactant production. By considering the importance of biosurfactants and their role in biodegradation of hydrocarbons and other industrial applications, the present investigation was conducted with the following objectives: production of biosurfactant by P. aeruginosa using waste motor lubricant oil and peanut oil cake, estimation of emulsification activity of the biosurfactant against hydrocarbons and influence of biosurfactant addition on biodegradation of crude oil.
Materials and Methods Organism Pseudomonas aeruginosa was isolated from sea water sample collected at Tuticorin harbor (08°450 N; 78°130 E) using Bushnell Haas agar supplemented with 0.1% (v/v) of crude oil and identified to species level by following Bergey’s Manual of Determinative Bacteriology [9]. Media and Growth Conditions Biosurfactant production was carried out in a 3 L laboratory fermentor with working volume of 2.1 L (Scigenics India Pvt. Ltd., Chennai). The strain was cultured in mineral medium (g/l, 1.0 K2HPO4, 0.2 MgSO47H2O, 0.05 FeSO4 7H2O, 0.1 CaCl22H2O, 0.001 Na2MoO42H2O, 30 NaCl). Optimization of culture conditions was carried out elsewhere [10]. The culture conditions are as follows—pH 8.0, temperature 38°C, salinity 30% (w/v) and 2.0% substrate concentration (waste motor lubricant oil/peanut oil cake) and 8.0 mg/l of dissolved oxygen (DO). Estimation of Growth and Biosurfactant Production Five ml samples of culture broth were collected at 12 h intervals for a period of 168 h. Biomass was estimated
31
gravimetrically, broth culture was filtered through a Millipore filter paper (0.45 lm) and dried at 80°C in hot air oven and weighed. Biomass was quoted in terms of mg/ml (dry weight). Biosurfactant concentration in the culture broth was estimated according to the procedure described by Li et al. [11] and the biosurfactant concentration was expressed as mg/ml. The culture broth was centrifuged at 6,000 rpm for 20 min at 4°C and extracted twice with chloroform and methanol (2:1 v/v). The solvents were removed by rotary evaporation and the residue was partially purified in silica gel (60–120 mesh) column eluted with chloroform and methanol ranging from 20:1 to 2:1 (v/v) in a gradient manner. The fractions were pooled and solvents were evaporated, resulting residue was dialyzed against distilled water and lyophilized. Estimation of Emulsification Activity Partially purified biosurfactant (5 mg) was dissolved in 5 ml of Tris buffer (pH 8.0) in 30 ml test tubes. Hydrocarbons like waste motor lubricant oil, crude oil, peanut oil, diesel, kerosene, naphthalene, anthracene and xylene were tested for emulsification activity. 5 mg of hydrocarbon was added to the above biosurfactant solution and shaken well for 20 min and the mixture was allowed to stand for 20 min. The optical density of the emulsified mixture was measured at 610 nm and the results were expressed as D610 [12]. Emulsification activity of the biosurfactant was compared with Triton X-100 (1 mg/ml) (Hi-Media, Laboratories, Mumbai, India), concentration and conditions for emulsification study were maintained similar to that of biosurfactant. Stability of the emulsion was measured by measuring the height of the emulsion layer and monitored for 24 h. Laboratory Scale Experiment on Biodegradation of Crude Oil with Biosurfactant This experiment was conducted to study the impact of the biosurfactant isolated from P. aeruginosa on biodegradation of crude oil in natural sea water. Crude oil used in this study was obtained from Chennai Refineries Limited, Chennai, India. Its specific gravity was reported by them as 0.844 at 25°C. Seventy-five liter plastic tanks were filled with 50 L of filtered and UV treated sea water with 30% salinity, and pH 8.0. The experiment was conducted with four different sets—(i) bacterial cells alone, (ii) with fertilizer (Urea and Dipotassium Phosphate 0.1% w/v) and cells, (iii) with cells and biosurfactants (0.1% w/v) and (iv) with cells, fertilizer and biosurfactant. 2.0% (w/v) of crude oil was added to the filtered sea water, inoculation was done with 24 h old culture (103–104 CFU/ml) at the rate of
123
32
1% (v/v) concentration. Continuous aeration was provided at the rate 1.5 L min-1 with an oil free aerator and the set up was maintained at room temperature for a period of 168 h. Biodegradation of crude oil was estimated fluorometrically as described in Intergovernmental Oceanographic Commission Manuals and Guide No. 11 [13]. An uninoculated control was maintained to assess the natural weathering of crude oil.
Characterization of Biosurfactant Biochemical Composition of Biosurfactant Carbohydrate content of the biosurfactant was determined by the phenol–sulfuric acid method [14] using D-glucose as a standard. Protein content was determined by the method of Lowry et al. [15] using bovine serum albumin as a standard and lipid content was estimated by following the procedure of Folch et al. [16]. Fourier Transform Infrared Spectroscopy Fourier transform infrared spectroscopy (FTIR) is most useful for identifying types of chemical bonds (functional groups), therefore can be used to elucidate some components of an unknown mixture. Freeze-dried crude biosurfactant (10 mg) was ground with 100 mg of KBr and pressed with 7,500 kg for 30 s to obtain translucent pellets. Infrared absorption spectra were recorded on a Thermo Niocolet, AVATAR 330 FTIR system with a spectral resolution and wave number accuracy of 4 and 0.01 cm-1, respectively. All measurements consisted of 500 scans, and a KBr pellet was used as background reference. Mass Spectrometric Analysis of Biosurfactant Biosurfactant was dissolved in methanol and mixed thoroughly. The mass spectrometric analysis of the biosurfactant was carried out in a LCQTM Quadrupole iontrap mass spectrometer (Finnigan MAT, San Jose, California, USA) utilising electrospray ionisation (ESI). Standard solutions and samples under investigation were infused into the mass spectrometer at a flow rate of 10 ll min-1. In the ESI, nitrogen and auxiliary gas flows were maintained at 50 and 5 ml/min respectively and refer to arbitrary values set by the software. The heated capillary temperature was 250°C and the spray voltage was set to 5 kV. Negative ion mode was used and scanning was done at 50–2,000 m/z range.
123
Indian J Microbiol (Jan–Mar 2011) 51(1):30–36
Results Estimation of Growth and Biosurfactant Production Physiological and biochemical characteristics of P. aeruginosa are illustrated in Table 1. Biosurfactant production was studied using 2.0% of waste motor oil and peanut oil cake. Figure 1 show the time-course of biosurfactant production by P. aeruginosa with waste motor oil as the substrate. Maximum biosurfactant concentration of 4.37 mg/ml occurred at 132 h of incubation, when the cells reached their early stationary phase. Maximum biomass was observed at 120 h (6.45 mg/ml). Biosurfactant production with peanut oil cake also showed similar trend, but with higher biosurfactant production (8.6 mg/ml) than waste motor lubricant oil (Fig. 1). Estimation of Emulsification Activity Biosurfactant isolated from P. aeruginosa and Triton X-100 showed maximum emulsification activity against waste motor lubricant oil. Emulsification of different hydrocarbons by the biosurfactant was in the order of waste motor lubricant oil [ crude oil [ peanut oil [ kerosene [ diesel [ xylene [ naphthalene [ anthracene (Table 2). The emulsion formed by the biosurfactant against each hydrocarbon was stable up to 48 h. Emulsification of eight different hydrocarbons by the biosurfactant reflects the
Table 1 Characteristics of P. aeruginosa Name of the test
Result
Gram reaction
-
Shape of the cell
Rod
Utilization of carbohydrates Adonitol
-
Glucose
?
Lactose
-
Trehalose
?
Xylose
?
Hydrolysis Starch
?
Gelatin
?
Fat
?
Catalase
?
Oxidase
?
Nitrate reduction
?
Litmus reaction
P
Decarboxylation Arginine ? Positive, - negative, P peptonization
?
Indian J Microbiol (Jan–Mar 2011) 51(1):30–36
BSW
10
BSP
BMW
14
BMP
9
12
8 10
7 6
8
5 6
4 3
4
Biomass Conc. (mg/ml)
Biosurfactant Conc. (mg/ml)
Fig. 1 Growth and biosurfactant production by P. aeruginosa in fermentor. BSW biosurfactant production using waste motor lubricant oil, BSP biosurfactant production using peanut oil cake, BMW biomass concentration with waste motor lubricant oil, BMP biomass concentration with peanut oil cake
33
2 2
1 0
0 0
12
24
36
48
60
72
84
96
108 120 132 144 156 168
Time (h)
possibility of its application against different hydrocarbon pollution. Laboratory Scale Experiment on Biodegradation of Crude Oil with Biosurfactant Biodegradation of crude oil in the laboratory scale experiment inferred that, maximum biodegradation was found with biosurfactant and fertilizer added set (88.2%) followed by biosurfactant (85%), fertilizer (71%) and 50.5% in normal setup. Characterization of Biosurfactant Biochemical composition of the biosurfactant revealed that, it is a mixture of lipid and protein with a combination of 49.8:50.2% respectively. FTIR analysis of the Table 2 Emulsification of hydrocarbons by biosurfactant isolated from P. aeruginosa and Triton X-100 Emulsification activity (D610) Biosurfactant
Triton X-100
Waste motor lubricant oil
2.01
1.93
Crude oil
1.97
1.85
Peanut oil
1.95
1.56
Kerosene
1.12
1.12
Diesel
0.90
0.94
Xylene
0.61
0.78
Naphthalene
0.51
0.63
Anthracene
0.45
0.58
*Experiment was carried out in duplicates and the mean values expressed as D610. Biosurfactant used in this assay was produced from peanut oil cake
biosurfactant showed that, wave numbers 3,422 and 3,246 cm-1 for N–H bonds indicated the presence of amine groups. C–H bonds of the CH3, CH2 and CH groups observed at wave numbers 2962, 2923, 2863, 1481 and 1425 cm-1 confirmed the presence of alkanes. The wave number 1,650 cm-1 (amide I bond) indicated the presence of peptide groups. The wave number 1,066 cm-1 indicated the presence of C–O bonds (Fig. 2). The above information from the respective wave numbers confirmed the lipopeptide nature of the biosurfactant. The mass spectrometric analysis of the biosurfactant also complements the biochemical and FTIR results that, the peaks observed at m/z = 1076.2, 1347.3, 1348.4 and 326.5, 413.3, 29.3 indicated the presence of protein and lipid moieties (Fig. 3).
Discussion This study revealed the possibility of biosurfactant production using cheaper carbon sources like waste motor lubricant oil and peanut oil cake. The possibility of biosurfactant production using cheaper carbon sources was already reported by earlier workers; Mercade et al. [17] used olive oil mill effluent, animal fat and frying oil by Deshpande and Daniels [18], molasses by Benincasa et al. [19], and starch-rich wastes by Nitschke and Pastore [20] supporting the present study on use of renewable carbon sources for biosurfactant production. Haba et al. [21] reported that, biosurfactant produced from firing oil have low emulsifying properties, where as biosurfactant produced in the present study using waste motor lubricant oil and peanut oil cake showed good emulsification activity against eight different hydrocarbons. Further, it encouraged the aim of the present study to produce biosurfactants from
123
34
Indian J Microbiol (Jan–Mar 2011) 51(1):30–36
Fig. 2 FTIR spectrum of biosurfactant produced by P. aeruginosa india pos mode4 #21 RT: 0.59 AV: 1 NL: 1.18E6 F: + c Full ms [ 250.00-2000.00] 326.5
100 95 90 85
1347.3
80 75
Relative Abundance
70 65 60 1348.4
55 50 45 40 663.4
35 30
1076.2
413.3 429.4
25
799.6
20 327.6 352.2
15 10
685.5 430.4
551.5 647.5 550.4
298.5
785.5 774.5
853.6 855.7
1014.1 927.5
1194.5
1269.1
1350.3
5 400
600
800
1000
1200
m/z
Fig. 3 Mass spectrum of biosurfactant produced by P. aeruginosa
123
1400
1489.8
1631.2 1659.2 1738.8
1600
1832.4
1800
1943.0
2000
Indian J Microbiol (Jan–Mar 2011) 51(1):30–36
cheaper carbon sources with high emulsification property. Parallel increase in biomass and biosurfactant were found from 12 to 120 h, but maximum biosurfactant concentration was found at 132 h. Higher concentration of biosurfactant even after the off set of biomass may be due to the release of cell bound biosurfactant at the early stationary phase (132 h), which leads to rise in extracellular biosurfactant concentration in the medium [22]. The emulsification activity of biosurfactant used in this study was higher than the emulsification activity recorded with Triton X-100 against waste motor lubricant oil, crude oil and peanut oil. Compare to Triton X-100 emulsification activity of the biosurfactant was low against kerosene, diesel, xylene, naphthalene and anthracene. However, while considering the advantages of biosurfactant over chemically synthesized surfactants, such as lower toxicity, biodegradability and ecological acceptability the possibility of replacing the chemical surfactant in oil pollution with biosurfactant is sought and need further research with different kind of experiments. Emulsification activity found with biosurfactant produced by P. aeruginosa in this study inferred that, biosurfactant produced with one carbon source like waste motor oil or peanut oil cake could be used to remediate different hydrocarbon pollution. Microbiological studies in laboratories, experimental field trials and cleanup operations following actual marine oil spill incidents, have demonstrated that bioremediation strategy based on the enhancement of oil biodegradation rates through nutrient addition was effective [23–27]. An understanding of the microbial hydrocarbon degradation process makes it possible to develop models for predicting the fate of hydrocarbon pollutants and to find strategies for incorporating microbial utilization of petroleum hydrocarbons in contaminated ecosystems [28]. In the present study, the attempt made on biodegradation of crude oil in a laboratory scale experimental setup revealed that maximum biodegradation rate was found with biosurfactant and fertilizer addition. Above information obtained in this study may be useful for the bioremediation of hydrocarbon polluted environments. Analysis of lipopeptide biosurfactant produced by P. putida using mass spectrometry was reported by Kuiper et al. [29] and their results were correlated with peaks observed the present study. Kalinovskaya et al. [30] also analyzed surfactin, a lipopeptide biosurfactant produced by Bacillus pumilus and got similar results. Chemically, biosurfactants produced with waste motor lubricant oil and peanut oil cake are similar. In conclusion, the present study is an attempt to find economically cheaper carbon sources for the large scale production of microbial biosurfactants. Results obtained in biosurfactant production with waste motor lubricant oil and peanut oil cake suggested the possibility of industrial production of biosurfactants using economically cheaper
35
carbon sources. Satisfactory emulsification activity of the biosurfactant against eight different hydrocarbons indicated its diverse applicability against different hydrocarbon pollution. Further purification, structural characterization of biosurfactant and genetic regulation of biosurfactant production are in progress. Acknowledgment The authors are thankful to the authorities of Annamalai University for providing the facilities and Department of Ocean Development and Council of Scientific and Industrial Research, Government of India for their financial support to carry out the work.
References 1. Rosenberg E (1986) Microbial biosurfactants. Crit Rev Biotechnol 3:109–132 2. Mercade ME, Manresa MA (1994) The use of agroindustrial byproducts for biosurfactant production. J Am Oil Chem Soc 71:61–64 3. Makkar RS, Cameotra SS (1999) Biosurfactant production by microorganisms on unconventional carbon sources. J Surfactants Deterg 2:237–242 4. Makkar RS, Cameotra SS (1997) Utilization of molasses for biosurfactant production by two Bacillus strains at thermophilic conditions. J Am Oil Chem Soc 74:887–889 5. Sheppard JD, Mulligan CN (1987) The production of surfactin by Bacillus subtilis grown on peat hydrolysate. Appl Microbiol Biotechnol 27:110–116 6. Fox SL, Bala GA (2000) Production of surfactant from Bacillus subtilis ATCC 21332 using potato substrates. Bioresour Technol 75:235–240 7. Besson F, Michel G (1992) Biosynthesis of iturin and surfactin by Bacillus subtilis. Evidence for amino acid activating enzymes. Biotechnol Lett 14:1013–1018 8. Cooper DG, Goldenberg BG (1987) Surface-active agents from two Bacillus species. Appl Environ Microbiol 53:224–229 9. Buchanan RE, Gibbons NE, Cowan ST, Holt TG, Liston J, Murry RG, Niven CF, Ravin AW, RY Stainer (1974) Bergey’s manual of determinative bacteriology. Williams and Wilkins Co., Baltimore, 1246 p 10. Thavasi R (2006) Biosurfactants from marine hydrocarbonoclastic bacteria and their application in marine oil pollution abatement. Ph.D. Thesis, Annamalai University, Chidambaram, 162 p 11. Li Z-Y, Lang S, Wagner F, Witte L, Wary V (1984) Formation and identification of interfacial-active glycolipids from resting microbial cells. Appl Environ Microbiol 48:610–617 12. Rosenberg E, Zuckerberg A, Rubinovitz C, Gutnick DL (1979) Emulsifier of Arthrobacter RAG-I: isolation and emulsifying properties. Appl Environ Microbiol 37:402–408 13. Intergovernmental Oceanographic Commission Manuals and Guide No. 11 (1982) The determination of petroleum hydrocarbons in sediments. UNESCO, Paris, 38 p 14. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356 15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275 16. Folch JM, Lees M, Stanly HS (1956) A simple method for the isolation and quantification of total lipids from animal tissues. J Biol Chem 226:497–509
123
36 17. Mercade ME, Manresa MA, Robert M, Espuny MJ, de Andres C, Guinea J (1993) Olive oil mill effluent (OOME). New substrate for biosurfactant production. Bioresour Technol 43:1–6 18. Deshpande M, Daniels L (1995) Evaluation of sophorolipid biosurfactant production by Candida bombicola using animal fat. Bioresour Technol 54:143–150 19. Benincasa M, Contiero J, Manresa MA, Moreaes IO (2002) Rhamnolipid production by Pseudomonas aeruginosa LBI growing on soap stock as the sole carbon source. J Food Eng 54:283–288 20. Nitschke M, Pastore GM (2004) Biosurfactant production by Bacillus subtilis using cassava processing effluent. Appl Biochem Biotechnol 112:163–172 21. Haba E, Espuny MJ, Busquets M, Manresa A (2000) Screening and production of rhamnolipids by Pseudomonas aeruginosa NCBI 40044 from waste frying oils. J Appl Microbiol 88:379–387 22. Goldman S, Shabtai Y, Rubinovitz C, Rosenberg E, Gutnick DL (1982) Emulsan in Acinetobacter calcoaceticus RAG-I: distribution of cell-free and cell associated cross reacting materials. Appl Environ Microbiol 44:165–170 23. Lee K, Tremblay GH, Cobanli SE (1995) Bioremediation of oiled beach sediments: assessment of inorganic and organic fertilizers. In: Proceedings of the 1995 oil spill conference. American Petroleum Institute, Washington, pp 107–113 24. Bragg JR, Prince E, Harner J, Atlas RM (1994) Effectiveness of bioremediation for the Exxon Valdez oil spill. Nature (London) 368:413–418
123
Indian J Microbiol (Jan–Mar 2011) 51(1):30–36 25. Fayad NM, Overton E (1995) A unique biodegradation pattern of the oil spilled during the 1991. Gulf War Mar Pollut Bull 30:239–246 26. Marty P, Martin Y (1996) Seed and feed strategy against oil spill in a marine environment: laboratory and simulated outdoor experiments with selected natural bacterial strains. J Mar Biotechnol 4:155–158 27. Kim S-J, Sohn JH, Sim DS, Kwon KK, Kim TH (1998) The effects of bioremediation on the oil degradation in oil polluted environments. In: Le Gal Y, Halvorson HO (eds) New developments in marine biotechnology. Plenum Press, New York, pp 181–188 28. Atlas RM (1981) Microbial degradation of petroleum hydrocarbons: an environmental perspective. Microbiol Rev 45:180–209 29. Kuiper I, Lagendijk EL, Pickford R, Derrick JP, Lamers GEM, Thomas-Oates JE, Lugtenberg BJJ, Bloemberg GV (2004) Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II. which inhibit biofilm formation and break down existing biofilms. Mol Microbiol 51:97–113 30. Kalinovskaya N, Kuznetsova T, Ya R, Yu M, Mil’grom E, Willis R, Wood A, Kurtz H, Carabedian C, Murphy P, Elyakov G (1995) Surfactin-like structures of five cyclic despsipeptises from the marine isolates of Bacillus pumilus Russ. Chem Bull (Engl Trans) 44:951
